Compositions and methods for passive optical barcoding for multiplexed assays

ABSTRACT

Compositions comprising multiple hydrogel particles having substantially the same diameter, but with each subgrouping of particles from the multiple hydrogel particles having different associated values for one or more passive optical properties that can be deconvoluted using cytometric instrumentation. Each hydrogel particle from the multiple hydrogel particles can be functionalized with a different biochemical or chemical target from a set of targets. A method of preparing hydrogel particles includes forming droplets and polymerizing the droplets, with optional functionalization.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.17/307,127, filed May 4, 2021 and titled “Compositions and Methods forPassive Optical Barcoding for Multiplexed Assays,” which claims priorityto and the benefit of U.S. Provisional Patent Application No.63/019,478, filed May 4, 2020 and titled “Compositions and Methods forPassive Optical Barcoding for Multiplexed Assays,” the disclosures ofeach of which are incorporated by reference herein in their entireties.

FIELD

The present disclosure relates to optically barcoding beads byengineering the passive optical properties of a hydrogel polymer, anduses thereof.

BACKGROUND

Flow cytometry and high-throughput cytometric analysis (e.g.,high-content imaging) are techniques that allow for the rapidseparation, counting, and characterization of individual cells, and areroutinely used in clinical and laboratory settings for a variety ofapplications. Cytometric devices are known in the art and includecommercially available devices for performing flow cytometry and FACS,hematology, and high-content imaging.

SUMMARY

In some embodiments, a composition comprises a hydrogel particle havingpassive optical properties (e.g., FSC and/or SSC) that are deliberatelyengineered, or “modulated,” without altering the size (e.g., thediameter) of the particle itself. The engineered hydrogels can then beused for multiplexing, using passive optical properties, optionally incombination with one or more additional properties, such asfluorescence, in order to perform a multiplexed assay (e.g., chemical orbiochemical) in a single reaction that can be deconvoluted based on thepassive optical properties of the individual bead populations.

In some embodiments, a method for producing a hydrogel particle includesforming droplets and polymerizing the droplets, with optionalfunctionalization. The method results in hydrogel particles havingsubstantially the same diameter, but different associated pre-determinedoptical properties (e.g., passive optical properties) that can bedeconvoluted using cytometric instrumentation.

In some embodiments, a method is provided for multiplexing assays. Themethod includes using a population of multiple hydrogel particles withunique passive optical properties in a single assay. Each hydrogelparticle from the multiple hydrogel particles having a unique associatedone or more biochemical targets. The population of multiple hydrogelparticles is assayed and the hydrogel particles and/or the biochemicaltargets are segregated based on their passive optical properties. Theresults of a multiplexed assay are then determined, based on the passiveoptical properties. Methods set forth herein allow for high-dimensional(>1) multiplexed assays to be performed in a single reaction, forexample using high throughput cytometric measurements.

BRIEF DESCRIPTION OF THE DRAWINGS

It is to be understood that the drawings primarily are for illustrativepurposes and are not intended to limit the scope of the subject matterdescribed herein.

FIG. 1 is a flow diagram showing a method for preparing hydrogelparticles, according to some embodiments.

FIG. 2 is a flow diagram showing a method for performing biochemicalmultiplexing, according to some embodiments.

FIG. 3A is a diagram illustrating the optical properties of a cell andthe disclosed hydrogel particles (A), according to some embodiments.

FIG. 3B is a diagram illustrating the optical properties of polystyrenebeads (B,C).

FIG. 4 is a diagram showing variables that can be tuned to encodespecific forward and side scatter “barcodes,” according to someembodiments.

FIG. 5 is a diagram showing particle formation in a microfluidicchannel, according to some embodiments.

FIG. 6 is a diagram showing an encoding scheme used to create apopulation of similarly-sized beads that can be de-multiplexed usingpassive optical properties, according to some embodiments.

FIG. 7A is a plot showing optical scatter data for multiple populationsof identically-sized particles that are distinguishable from one anotherbased on their distinct optical scatter properties. FIG. 7B, bycontrast, includes fluorescence plots for each of the multiple particlepopulations of FIG. 7A, showing that the particle populations of FIG. 7Bcannot be distinguished from one another based on fluorescence alone.FIGS. 7A-7B demonstrate the ability to perform biochemical multiplexingand demultiplexing using passive optical properties as a primarydeconvolution variable, according to some embodiments.

FIG. 8A is a plot of white blood cell counts for an example populationof lysed whole blood, and FIG. 8B is a plot of counts for hydrogelparticles with tuned passive optical properties, according to anexperimental example as an elegant demonstration of optical tuning for abiologically-relevant target population.

FIG. 9A is a plot of side scatter data for hydrogel particles with tunedpassive optical properties, and FIG. 9B is a plot of forward scatterdata for the hydrogel particles of FIG. 9A. FIGS. 9A-9B show that sidescatter can be tuned independently of forward scatter, according to anexperimental example.

FIG. 10A is a plot of side scatter versus forward scatter for a firstacrylamide:bis-acrylamide ratio. FIG. 10B is a plot of side scatterversus forward scatter for a second acrylamide:bis-acrylamide ratiogreater than the first acrylamide:bis-acrylamide ratio. FIG. 10C is aplot of side scatter versus forward scatter for thirdacrylamide:bis-acrylamide ratio greater than the secondacrylamide:bis-acrylamide ratio. FIGS. 10A-10C show that forward scatterincreases with negligible impact on side scatter, with increasingpercentages of acrylamide:bis-acrylamide, according to an experimentalexample.

DETAILED DESCRIPTION

Flow cytometry and high-throughput cytometric analysis can also be usedto assay beads (e.g., for biochemical measurements). In some suchimplementations, a beam of light is directed onto a focused stream ofliquid containing the beads. Multiple detectors are then aimed at thepoint where the stream passes through the light beam, with one detectorin line with the light beam (e.g., to detect forward scatter (“FSC”))and several detectors perpendicular to the light beam (e.g., to detectside scatter (“SSC”)). FSC and SSC measurements are typically referredto as “passive optical properties.” For particles such as cells (e.g.,human cells), FSC typically correlates with cell volume, while SSCtypically correlates with the inner complexity, or granularity, of theparticle (e.g., shape of the nucleus, the amount and type of cytoplasmicgranules, or the membrane roughness). As a result of these correlations,different specific cell types can exhibit different FSC and SSC, suchthat cell types can be distinguished from one another based on theirpassive optical properties in flow cytometry. These measurements—FSC andSSC—form the basis of cytometric analysis in clinical and researchsettings. Most synthetic or polymer products used in such cellularanalyses are made of (or substantially comprise) polystyrene orlatex—opaque polymers that generally have fixed FSC and SSC values basedon the diameter of the particle itself. As such, polystyrene particlesof the same diameter generally cannot be distinguished from one anotherbased on passive optical properties (FSC and SSC) alone.

To distinguish subpopulations of similarly-sized polystyrene particlesfrom one another, a fluorophore can be added to the particle, allowingfor multiplexed (e.g., multi-color) assays. By combining differentfluorophores at different concentrations into a single bead, one cancreate a unique identifier that allows the bead to be distinguished fromwithin a population of distinctly stained beads. When combined withunique assay targets, fluorescently barcoded bead populations such canfacilitate the simultaneous assaying of multiple targets (referred toherein as “biochemical multiplexing”). Known products, such as Luminexbeads, however, are limited to fluorescent multiplexing because they aremade of polystyrene, which as noted above, has fixed passive opticalproperties. In addition to the material limitations of existing productsmade of polystyrene, the instruments used to measure such beadstypically have a fixed and limited number of fluorescence detectors,placing a limit on the number of dimensions/targets afluorescence-driven multiplexing strategy can address. Modernbiochemical assays stand to benefit from additional dimensions ofmultiplexing, but are limited by instrument detector availability. Assuch, there is a need for a product that allows for additional andorthogonal dimensions of multiplexing using passive optical properties.Embodiments set forth herein address this need through passive opticalbarcoding of hydrogel substrates.

Some known products, such as LEGENDplex (BioLegend), use particles ofdifferent sizes to perform >1 plex assays. Although the populations ofbeads in such products may be distinguishable via their passive opticalproperties (LEGENDplex, BioLegend), they inherently possess differentsurface areas, hydrodynamic, and biochemical properties (e.g.concentrations of analytes) as a result of the size differences, leadingto suboptimal assay performance and non-quantitative measurements.

General Overview

In view of the fluidic conditions that exist within flow cytometers andhigh-content imaging systems, particles used for biochemical assays orcalibration typically fall within a limited size range to avoid particlesettling and related clogging of the fluidics (which can occur withlarger particles), and/or to avoid particles floating to the surface ofa liquid suspension (which can occur with small particles, makingeffective sampling challenging). This size restriction limits theforward scattering range that polystyrene particles can elicit. Unlikepolystyrene particles, hydrogel particles disclosed herein can exhibit avariety of different optical scatter properties while remaining a fixeddiameter, thereby facilitating the optimization of fluidic propertiesand the introduction of additional dimensions of multiplexing.

FIG. 1 is a flow diagram showing a method for preparing hydrogelparticles, according to some embodiments. As shown in FIG. 1 , themethod 100 includes droplet formation at 110 (e.g., to produce apolydisperse plurality of droplets or a monodisperse plurality ofdroplets, as described herein). At 112, one or more surfactants areoptionally added to the droplets, and at 114, one or more co-monomersare added to the droplets. The droplets are then polymerized, at 116, toform the hydrogel particles, which are optionally subsequentlyfunctionalized, at 118 (e.g., with one or more chemical side groups orfluorescent dyes, as discussed further below).

FIG. 2 is a flow diagram showing a method for performing biochemicalmultiplexing, according to some embodiments. As shown in FIG. 2 , themethod 200 includes providing a plurality of engineered hydrogelparticles, at 220, each engineered hydrogel particle from the pluralityof engineered hydrogel particles having its own unique passive opticalproperties. The engineered hydrogel particles can be transparent orsemi-transparent. At 222, an assay is prepared, including the engineeredhydrogel particles and at least one biochemical target. At 224, one ormore passive optical properties of the engineered hydrogel particles aremeasured. Based on these measurements, the engineered hydrogel particlesand/or the at least one biochemical target can be segregated, at 226,and/or assay results can be determined, at 228.

FIG. 3A is a diagram illustrating the optical properties of a cell andthe disclosed hydrogel particles, according to some embodiments. Asshown in FIG. 3A, the engineered hydrogels described herein aresemi-transparent, allowing internal features thereof (i.e., cellularcomplexity) to be resolved using a side scatter (SSC) detector. Bycontrast, FIG. 3B is a diagram illustrating the optical properties ofpolystyrene beads. In contrast with FIG. 3A, the polystyrene beads ofFIG. 3B are opaque and have a fixed SSC that is determined by theirdiameter and that is not impacted by (i.e., does not change based on)internal features thereof. As such, polystyrene particles are of limitedutility in the two most important passive optical measurements used inflow cytometry: FSC and SSC, which measure the size and complexity ofthe target, respectively. Due to these limitations of polystyrene, usersmust typically rely on fluorescence alone for multiplexedimmunophenotyping experiments.

In some embodiments set forth herein, a composition comprises a hydrogelparticle that is engineered to have passive optical properties that canbe distinguished from the optical properties of other particles (e.g.,hydrogel particles) of the same diameter, using FSC and SSC alone. Theinventors have unexpectedly discovered that optical properties of ahydrogel particle can be independently modulated by altering thecomposition of the hydrogel particle. For example, SSC can be modulatedwithout substantially affecting FSC, and vice versa (i.e., FSC can bemodulated without substantially affecting SSC). Furthermore, the opticalproperties (e.g., refractive index) of hydrogel particles can be tunedwithout having a substantial effect on density or on the size of theparticles themselves. This is a surprising and useful feature, as theseproperties allow for multiple particles of the same size to be “encoded”with specific FSC/SSC ratios, and later deconvoluted using detectorssuch as the detectors found on all cytometric instrumentation, includinglow-cost instrumentation without fluorescent measurement capabilities.

In some embodiments, a method of producing a hydrogel particle resultsin the hydrogel particle having pre-determined optical properties. Insome embodiments, a method of multiplexing assays includes using aplurality (or “population”) of hydrogel particles with unique passiveoptical properties, each having unique biochemical targets, in a singleassay. One or more passive optical properties of the population aremeasured, and the population and/or the biochemical targets aresegregated based on the measured passive optical properties. The resultsof the multiplexed assay can be generated based on the measured passiveoptical properties. The foregoing procedure and the engineered nature ofthe hydrogel particles facilitate performing high-dimensionalmultiplexed assays in a single reaction, and the segregation of thehydrogel particles and/or the biochemical targets using high throughputcytometric measurements.

Hydrogels

Hydrogel particles set forth herein comprise a hydrogel. A hydrogel is amaterial comprising a macromolecular three-dimensional network thatallows it to swell when in the presence of water, and to shrink in theabsence of (or by reduction of the amount of) water, but not to dissolvein water. The swelling, i.e., the absorption of water, is a consequenceof the presence of hydrophilic functional groups attached to ordispersed within the macromolecular network. Cross-links betweenadjacent macromolecules result in the aqueous insolubility of thesehydrogels. The cross-links may be due to chemical bonding (i.e.,covalent bonds) or physical bonding (i.e., Van Der Waal forces,hydrogen-bonding, ionic forces, etc.). While some in the polymerindustry may regard the macromolecular material(s) set forth hereinto bea “xerogel” in the dry state and a “hydrogel” in the hydrated state, forpurposes of the present disclosure, the term “hydrogel” will refer tothe macromolecular material whether dehydrated or hydrated. Acharacteristic of a hydrogel that is of particular value is that thematerial retains its general shape/morphology, whether dehydrated orhydrated. Thus, if the hydrogel has an approximately spherical shape inthe dehydrated condition, it will be spherical in the hydratedcondition.

Hydrogels set forth herein can comprise greater than about 30%, greaterthan about 40%, greater than about 50%, greater than about 55%, greaterthan about 60%, greater than about 65%, greater than about 70%, greaterthan about 75%, greater than about 80%, or greater than about 85% water.

In some embodiments, synthetic hydrogels can be prepared by polymerizinga monomeric material (“hydrogel monomer”) to form a backbone, andcross-linking the backbone with a cross-linking agent. Suitable hydrogelmonomers include (but are not limited to) the following: lactic acid,glycolic acid, acrylic acid, 1-hydroxyethyl methacrylate, ethylmethacrylate, propylene glycol methacrylate, acrylamide,N-vinylpyrrolidone, methyl methacrylate, glycidyl methacrylate, glycolmethacrylate, ethylene glycol, fumaric acid, and the like. Suitablecross-linking agents include (but are not limited to) tetraethyleneglycol dimethacrylate and N,N′-15 methylenebisacrylamide. In someembodiments, a hydrogel particle is produced via the polymerization ofacrylamide.

In some embodiments, a hydrogel comprises a mixture of at least onemonofunctional monomer and at least one bifunctional monomer.

A monofunctional monomer can be a monofunctional acrylic monomer.Non-limiting examples of monofunctional acrylic monomers are acrylamide;methacrylamide; N-alkylacrylamides such as N-ethylacrylamide,N-isopropylacrylamide or N-tert-butylacrylamide; N-alkylmethacrylamidessuch as N-ethylmethacrylamide or N-isopropylmethacrylamide;N,N-dialkylacrylamides such as N,N-dimethylacrylamide andN,N-diethyl-acrylamide; N-[(dialkylamino)alkyl]acrylamides such as N-[3dimethylamino)propyl]acrylamide or N-[3-(diethylamino)propyl]acrylamide;N-[(dialkylamino)alkyl]methacrylamides such asN-[3-dimethylamino)propyl]methacrylamide orN-[3-(diethylamino)propyl]methacrylamide; (dialkylamino)alkyl acrylatessuch as 2-(dimethylamino)ethyl acrylate, 2-(dimethylamino)propylacrylate, or 2-(diethylamino)ethyl acrylates; and (dialkylamino)alkylmethacrylates such as 2-(dimethylamino)ethyl methacrylate.

A bifunctional monomer is any monomer that can polymerize with amonofunctional monomer of the disclosure to form a hydrogel as describedherein that further contains a second functional group that canparticipate in a second reaction, e.g., conjugation of a fluorophore.

In some embodiments, a bifunctional monomer is selected from the groupconsisting of: allyl alcohol, allyl isothiocyanate, allyl chloride, andallyl maleimide.

A bifunctional monomer can be a bifunctional acrylic monomer.Non-limiting examples of bifunctional acrylic monomers areN,N′-methylenebisacrylamide, N,N′-methylenebismethacrylamide,N,N′-ethylenebiscrylamide, N,N′-ethylenebis-methacrylamide,N,N′propylenebisacrylamide andN,N′-(1,2-dihydroxyethylene)bisacrylamide.

Higher-order branched chain and linear co-monomers can be substituted inthe polymer mix to adjust the refractive index while maintaining polymerdensity, as described in U.S. Pat. No. 6,657,030, titled “HighRefractive Index Hydrogel Compositions for Ophthalmic Implants,” thecontent of which is incorporated herein by reference in its entirety,for all purposes.

In some embodiments, a hydrogel comprises a molecule that modulates theoptical properties of the hydrogel. Molecules capable of alteringoptical properties of a hydrogel are discussed further below.

Naturally-occurring hydrogels useful for embodiments set forth hereininclude various polysaccharides available or derived from naturalsources, such as plants, algae, fungi, yeasts, marine invertebrates andarthropods. Non-limiting examples of polysaccharides suitable for use inthe embodiments set forth herein include (but are not limited to)agarose, dextrans, chitin, cellulose-based compounds, starch,derivatized starch, and the like. Such polysaccharides may includepluralities of repeating glucose units as a major portion of thepolysaccharide backbone.

Polymerization of a hydrogel can be initiated by a persulfate. Thepersulfate can be any water-soluble persulfate. Non-limiting examples ofwater soluble persulfates are ammonium persulfate and alkali metalpersulfates. Alkali metals include lithium, sodium and potassium. Insome preferred embodiments, the persulfate is ammonium persulfate orpotassium persulfate.

Polymerization of a hydrogel can be accelerated by an accelerant. Theaccelerant can be a tertiary amine. The tertiary amine can be anywater-soluble tertiary amine. Preferably, the tertiary amine isN,N,N′,N′tetramethylethylenediamine (TEMED) or3-dimethylamino)propionitrile.

FIG. 4 is a diagram showing variables that can be tuned to encodespecific passive optical (e.g., FSC and/or SSC) “barcodes” on/ingroupings of hydrogel particles, according to some embodiments. As shownin the top row (1) of FIG. 4 , adjustments in a monomer/co-monomer ratioand crosslinking density can lead to changes in the refractive index ofthe hydrogel particles (e.g., increasing from n, to 2*n, to 3*n withincreasing ratio of monomer::co-monomer). The middle row (2) of FIG. 4shows that adjustments in nanoparticle composition and concentration canadjust the SSC of the hydrogel particles (e.g., increasing SSC withincreased concentration of nanoparticles), and the bottom row (3) ofFIG. 4 shows that functionalization of the hydrogel particles withchemical side groups can result in precise, stoichiometric ratios ofsecondary labels (e.g. fluorophores, proteins, antigens, antibodies) onthe hydrogel particles. This feature enables quantitative meanfluorescence intensity (MFI) to be controlled on the particle, a uniquefeature of the particles described herein.

FIG. 5 is a diagram showing particle formation in an oil-filledmicrofluidic channel, according to some embodiments.

Hydrogel Particles

In some embodiments, a hydrogel particle includes a hydrogel and isproduced by polymerizing a droplet (see the discussion of “dropformation” in relation to FIG. 5 ). Microfluidic methods of producing aplurality of droplets, including fluidic and rigidified droplets, caninclude one or more methods described in U.S. Patent ApplicationPublication No. 2011/0218123, titled “Creation of Libraries of Dropletsand Related Species,” and U.S. Pat. No. 7,294,503, titled“Microfabricated Crossflow Devices and Methods,” the contents of each ofwhich are incorporated herein by reference in their entireties, for allpurposes. Such methods provide for the generation of a plurality ofdroplets, each droplet from the plurality of droplets containing a firstfluid that is substantially surrounded by a second fluid, where thefirst fluid and the second fluid are substantially immiscible (e.g.,droplets containing an aqueous-based liquid being substantiallysurrounded by an oil based liquid). In other embodiments, particles canbe created via precipitation polymerization, or membrane emulsification.

A plurality of fluidic droplets (e.g., prepared using a microfluidicdevice) may be polydisperse (e.g., having a range of different sizes),or in some cases, the fluidic droplets may be monodisperse orsubstantially monodisperse, e.g., having a homogenous distribution ofdiameters, for instance, such that no more than about 10%, about 5%,about 3%, about 1%, about 0.03%, or about 0.01% of the droplets have anaverage diameter greater than about 10%, about 5%, about 3%, about 1%,about 0.03%, or about 0.01% of the average diameter. The averagediameter of a population of droplets, as used herein, refers to thearithmetic average of the diameters of the droplets.

In some embodiments, a population of hydrogel particles includes aplurality of hydrogel particles, and the population of hydrogelparticles is substantially monodisperse.

The term “microfluidic” refers to a device, apparatus or systemincluding at least one fluid channel having a cross-sectional dimensionof less than 1 mm, and a ratio of length to largest cross-sectionaldimension perpendicular to the channel of at least about 3:1. Amicrofluidic device comprising a microfluidic channel is especially wellsuited to preparing a plurality of monodisperse droplets. Cross-flowmembrane emulsification and precipitation polymerization are othersuitable methods for generating a plurality of monodispersed droplets.

Non-limiting examples of microfluidic systems that may be used with thepresent invention include those disclosed in U.S. Patent ApplicationPublication No. 2006/0163385 (“Forming and Control of Fluidic Species”),U.S. Patent Application Publication No. 2005/0172476 (“Method andApparatus for Fluid Dispersion”), U.S. Patent Application PublicationNo. 2007/000342 (“Electronic Control of Fluidic Species”), InternationalPatent Application Publication No. WO 2006/096571 (“Method and Apparatusfor Forming Multiple Emulsions”), U.S. Patent Application PublicationNo. 2007/0054119 (“Systems and Methods of Forming Particles”),International Patent Application Publication No. WO 2008/121342(“Emulsions and Techniques for Formation”), and International PatentApplication Publication No. WO 2006/078841 (“Systems and Methods forForming Fluidic Droplets Encapsulated in Particles Such as ColloidalParticles”), the entire contents of each of which are incorporatedherein by reference in their entireties, for all purposes.

Droplet size can be related to microfluidic channel size, pore size (inthe case of membrane emulsification) and/or flow rate. The microfluidicchannel can be any of a variety of sizes, for example, having a largestdimension perpendicular to fluid flow of less than about 5 mm, or lessthan about 2 mm, or less than about 1 mm, or less than about 500 μm,less than about 200 μm, less than about 100 μm, less than about 60 μm,less than about 50 μm, less than about 40 μm, less than about 30 μm,less than about 25 μm, less than about 10 μm, less than about 3 μm, lessthan about 1 μm, less than about 300 nm, less than about 100 nm, lessthan about 30 nm, or less than about 10 nm.

Droplet size can be tuned by adjusting the relative flow rates. In someembodiments, drop diameters are equivalent to the width of the channel,or within about 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or100% the width of the channel.

In some embodiments, the dimensions of a hydrogel particle aresubstantially similar to the dimensions of a droplet from which it wasformed. For example, in some such embodiments, a hydrogel particle has adiameter of less than about 1 μm, less than about 2 μm, less than about5 μm, less than about 10 μm, less than about 15 μm, less than about 20μm, less than about 25 μm, less than about 30 μm, less than about 35 μm,less than about 40 μm, less than about 45 μm, less than about 50 μm,less than about 60 μm, less than about 70 μm, less than about 80 μm,less than about 90 μm, less than about 100 μm, less than about 120 μm,less than about 150 μm, less than about 200 μm, less than about 250 μm,less than about 300 μm, less than about 350 μm, less than about 400 μm,less than about 450 μm, less than about 500 μm, less than about 600 μm,less than about 800 μm, or less than 1000 μm in diameter. In someembodiments, a hydrogel particle has a diameter of greater than about 1μm, greater than about 2 μm, greater than about 5 μm, greater than about10 μm, greater than about 15 μm, greater than about 20 μm, greater thanabout 25 μm, greater than about 30 μm, greater than about 35 μm, greaterthan about 40 μm, greater than about 45 μm, greater than about 50 μm,greater than about 60 μm, greater than about 70 μm, greater than about80 μm, greater than about 90 μm, greater than about 100 μm, greater thanabout 120 μm, greater than about 150 μm, greater than about 200 μm,greater than about 250 μm, greater than about 300 μm, greater than about350 μm, greater than about 400 μm, greater than about 450 μm, greaterthan about 500 μm, greater than about 600 μm, greater than about 800 μm,or greater than 1000 μm in diameter. In typical embodiments, a hydrogelparticle has a diameter in the range of 5 μm to 100 μm.

In some embodiments, one or more hydrogel particles are spherical inshape.

In some embodiments, one or more hydrogel particles have materialmodulus properties (e.g., elasticity) more closely resembling thecorresponding material modulus properties of a target cell (e.g., ahuman target cell), as compared with the corresponding material modulusproperties of a polystyrene bead having the same diameter as thehydrogel particle.

In some embodiments, one or more hydrogel particles do not compriseagarose.

Optical properties

Passive Optical Properties

The three primary modes of deconvolution for flow cytometry are the twopassive optical properties of a particle (forward scattering, FSC,corresponding to the refractive index, or RI; and side scattering, SSC),and the biomarkers present on the surface of a given cell type, whichare typically measured via fluorescence. Compositions set forth herein,which allow these properties to be rationally engineered, allow forassay multiplexing, or measuring more than one target (e.g., a cell, amolecule, a biochemical target, etc.) at a time, through deconvolution.

In some embodiments, the refractive index (RI) of one or more hydrogelparticles is greater than about 1.10, greater than about 1.15, greaterthan about 1.20, greater than about 1.25, greater than about 1.30,greater than about 1.35, greater than about 1.40, greater than about1.45, greater than about 1.50, greater than about 1.55, greater thanabout 1.60, greater than about 1.65, greater than about 1.70, greaterthan about 1.75, greater than about 1.80, greater than about 1.85,greater than about 1.90, greater than about 1.95, greater than about2.00, greater than about 2.10, greater than about 2.20, greater thanabout 2.30, greater than about 2.40, greater than about 2.50, greaterthan about 2.60, greater than about 2.70, greater than about 2.80, orgreater than about 2.90.

In some embodiments, the RI of one or more hydrogel particles is lessthan about 1.10, less than about 1.15, less than about 1.20, less thanabout 1.25, less than about 1.30, less than about 1.35, less than about1.40, less than about 1.45, less than about 1.50, less than about 1.55,less than about 1.60, less than about 1.65, less than about 1.70, lessthan about 1.75, less than about 1.80, less than about 1.85, less thanabout 1.90, less than about 1.95, less than about 2.00, less than about2.10, less than about 2.20, less than about 2.30, less than about 2.40,less than about 2.50, less than about 2.60, less than about 2.70, lessthan about 2.80, or less than about 2.90

In some embodiments, the SSC of one or more hydrogel particles can beany value within the full range of possible values as measured by acytometric device.

In some embodiments, the FSC of one or more hydrogel particles can beany value within the full range of possible values as measured by acytometric device.

In some embodiments, the FSC of one or more hydrogel particles can betuned by incorporating a high-refractive index molecule in the hydrogel.Preferred high-refractive index molecules include (but are not limitedto) colloidal silica, alkyl acrylate and alkyl methacrylate. Thus, insome embodiments, one or more hydrogel particles include alkyl acrylateand/or alkyl methacrylate. Alkyl acrylates or Alkyl methacrylates cancontain 1 to 18, 1 to 8, or 2 to 8, carbon atoms in the alkyl group,such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl ortert-butyl, 2-ethylhexyl, heptyl or octyl groups. The alkyl group may bebranched or linear. High-refractive index molecules can also includevinylarenes such as styrene and methylstyrene, optionally substituted onthe aromatic ring with an alkyl group, such as methyl, ethyl ortert-butyl, or with a halogen, such as chlorostyrene.

In some embodiments, the FSC of one or more hydrogel particles ismodulated by adjusting the water content present during hydrogelformation. In other embodiments, the FSC of one or more hydrogelparticles is modulated by adjusting crosslinking density of thehydrogel. Alternatively or in addition, the FSC of one or more hydrogelparticles can be related to particle volume, and thus can be modulatedby altering particle diameter, as described herein.

In some embodiments, the SSC of one or more hydrogel particles can beengineered by encapsulating nanoparticles within hydrogels. In someembodiments, a hydrogel particle comprises one or more types ofnanoparticles, for example selected from the group consisting of:polymethyl methacrylate (PMMA) nanoparticles, polystyrene (PS)nanoparticles, and silica nanoparticles.

Functionalization of Hydrogel Particles

In some embodiments, in addition to having specific and engineeredpassive optical properties, hydrogel particles set forth herein can befunctionalized, allowing them to mimic the fluorescent properties oflabeled cells. In some embodiments, a hydrogel particle comprises abifunctional monomer, and functionalization of the hydrogel particleoccurs via the bifunctional monomer. In some embodiments, afunctionalized hydrogel particle comprises a free amine group. In otherembodiments, the hydrogel can be functionalized with a protein orpeptide which allows for secondary labeling using a reagent, includingbut not limited to an antibody.

A hydrogel particle can be functionalized with any fluorescent dye,including any of the fluorescent dyes listed in The MolecularProbes®Handbook—A Guide to Fluorescent Probes and Labeling Technologies, thecontent of which is incorporated herein by reference in its entirety,for all purposes. Functionalization can be mediated by a compoundcomprising a free amine group, e.g., allylamine, which can beincorporated into a hydrogel particle during the formation process.

Non-limiting examples of suitable fluorescent dyes include:6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein succinimidyl ester;5-(and-6)-carboxyeosin; 5-carboxyfluorescein; 6-carboxyfluorescein;5-(and-6)-carboxyfluorescein;5-carboxyfluorescein-bis-(5-carboxymethoxy-2-nitrobenzyl)ether,-alanine-carboxamide, or succinimidyl ester; 5-carboxyfluoresceinsuccinimidyl ester; 6-carboxyfluorescein succinimidyl ester;5-(and-6)-carboxyfluorescein succinimidyl ester;5-(4,6-dichlorotriazinyl) aminofluorescein; 2′,7′-difluorofluorescein;eosin-5-isothiocyanate; erythrosin 5-isothiocyanate;6-(fluorescein-5-carboxamido)hexanoic acid or succinimidyl ester;6-(fluorescein-5-(and-6)-carboxamido)hexanoic acid or succinimidylester; fluorescein-5-EX succinimidyl ester;fluorescein-5-isothiocyanate; fluorescein-6-isothiocyanate; OregonGreen®488 carboxylic acid, or succinimidyl ester; Oregon Green® 488isothiocyanate; Oregon Green® 488-X succinimidyl ester; Oregon Green®500 carboxylic acid; Oregon Green® 500 carboxylic acid, succinimidylester or triethylammonium salt; Oregon Green® 514 carboxylic acid;Oregon Green® 514 carboxylic acid or succinimidyl ester; RhodamineGreen™carboxylic acid, succinimidyl ester or hydrochloride; Rhodamine Green™carboxylic acid, trifluoroacetamide or succinimidyl ester; RhodamineGreen™-X succinimidyl ester or hydrochloride; RhodolGreen™ carboxylicacid, N,0-bis-(trifluoroacetyl) or succinimidyl ester;bis-(4-carboxypiperidinyl) sulfonerhodamine or di(succinimidyl ester);5-(and-6)carboxynaphtho fluorescein, 5-(and-6)carboxynaphtho fluoresceinsuccinimidyl ester; 5-carboxyrhodamine 6G hydrochloride;6-carboxyrhodamine 6G hydrochloride, 5-carboxyrhodamine 6G succinimidylester; 6-carboxyrhodamine 6G succinimidyl ester;5-(and-6)-carboxyrhodamine 6G succinimidyl ester;5-carboxy-2′,4′,5′,7′-tetrabromosulfonefluorescein succinimidyl esterorbis-(diisopropylethylammonium) salt; 5-carboxytetramethylrhodamine;6-carboxytetramethylrhodamine; 5-(and-6)-carboxytetramethylrhodamine;5-carboxytetramethylrhodamine succinimidyl ester;6-carboxytetramethylrhodamine succinimidyl ester;5-(and-6)-carboxytetramethylrhodamine succinimidyl ester;6-carboxy-X-rhodamine; 5-carboxy-X-rhodamine succinimidyl ester;6-carboxy-Xrhodamine succinimidyl ester;5-(and-6)-carboxy-Xrhodaminesuccinimidyl ester; 5-carboxy-X-rhodaminetriethylammonium salt; Lissamine™ rhodamine B sulfonyl chloride;malachite green; isothiocyanate; NANOGOLD® mono(sulfosuccinimidylester); QSY® 21 carboxylic acid or succinimidyl ester; QSY® 7 carboxylicacid or succinimidyl ester; Rhodamine Red™-X succinimidyl ester;6-(tetramethylrhodamine-5-(and-6)-carboxamido)hexanoic acid;succinimidyl ester; tetramethylrhodamine-5-isothiocyanate;tetramethylrhodamine-6-isothiocyanate;tetramethylrhodamine-5-(and-6)-isothiocyanate; Texas Red® sulfonyl;Texas Red® sulfonyl chloride; Texas Red®-X STP ester or sodium salt;Texas Red®-X succinimidyl ester; Texas Red®-X succinimidyl ester; andX-rhodamine-5-(and-6)-isothiocyanate.

Other examples of fluorescent dyes include BODIPY® dyes commerciallyavailable from Invitrogen, including, but not limited to BODIPY® FL;BODIPY® TMR STP ester; BODIPY® TR-X STP ester; BODIPY® 630/650-XSTPester; BODIPY® 650/665-X STP ester;6-dibromo-4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoicacid; 4,4-difluoro-5,7-dimethyl-4-bora3a,4a-diaza-s-indacene-3-pentanoicacid succinimidyl ester;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionic acid;4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acidsuccinimidyl ester;4,4difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionic acid;sulfosuccinimidyl ester or sodium salt;6-((4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3propionyl)amino)hexanoicacid;6-((4,4-difluoro-5,7dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid or succinimidyl ester;N-(4,4-difluoro5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)cysteicacid, succinimidyl ester or triethylammonium salt;6-4,4-difluoro-1,3-dimethyl-5-(4-methoxyphenyl)-4-bora3a,4a4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionicacid; 4,4-difluoro-5,7-diphenyl-4-bora3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;4,4-difluoro-5-phenyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;succinimidyl ester;6-((4,4-difluoro-5-phenyl-4bora-3a,4a-diaza-s-indacene-3-propionyl)amino)hexanoicacid or succinimidyl ester;4,4-difluoro-5-(4-phenyl-1,3butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;6-(((4,4-difluoro-5-(2-pyrrolyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester;4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;4,4-difluoro-5-styryl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid;succinimidyl ester;4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-propionicacid;4,4-difluoro-1,3,5,7-tetramethyl-4bora-3a,4a-diaza-s-indacene-8-propionicacid succinimidyl ester;4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-propionicacid succinimidyl ester;6-(((4-(4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)phenoxy)acetyl)amino)hexanoicacid or succinimidyl ester; and6-(((4,4-difluoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-yl)styryloxy)acetyl)aminohexanoicacid or succinimidyl ester.

Fluorescent dyes can also include, for example, Alexa fluor dyescommercially available from Invitrogen, including but not limited toAlexa Fluor® 350 carboxylic acid; Alexa Fluor® 430 carboxylic acid;Alexa Fluor® 488 carboxylic acid; Alexa Fluor® 532 carboxylic acid;Alexa Fluor® 546 carboxylic acid; Alexa Fluor® 555 carboxylic acid;Alexa Fluor® 568 carboxylic acid; Alexa Fluor® 594 carboxylic acid;Alexa Fluor® 633 carboxylic acid; Alexa Fluor® 647 carboxylic acid;Alexa Fluor® 660 carboxylic acid; and Alexa Fluor® 680 carboxylic acid.Suitable fluorescent dyes can also include, for example, cyanine dyescommercially available from Amersham-Pharmacia Biotech, including, butnot limited to Cy3 NHS ester; Cy5 NHS ester; Cy5.5 NHSester; and Cy7 NHSester.

EXAMPLES Example I Generation of Hydrogel Particles

Photomasks for UV lithography were sourced from FineLine Imaging, Inc.and were designed using AutoCad (AutoDesk, Inc.). SU-8 photo resist(Microchem, Inc.) was photo cross-linked on 4″ silicon wafers using acollimated UV light source (OAI, Inc.) to create masters formicrofluidic device fabrication. PDMS (polydimethylsiloxane, SigmaAldrich, Inc.) was prepared and formed using soft lithography andmicrofluidic device fabrication methods (See, e.g., McDonald J C, etal., 2000, Electrophoresis 21:27-40, the contents of which areincorporated herein by reference in their entirety, for all purposes).

Droplets were formed using flow-focusing geometry, in which two oilchannels focus a central stream of aqueous monomer solution to break offdroplets in a water-in-oil emulsion. A fluorocarbon-oil (Novec 7500 3M,Inc.) was used as the outer, continuous phase liquid for dropletformation. To stabilize droplets before polymerization, a surfactant wasadded at 0.5% w/w to the oil phase (ammonium carboxylate salt of Krytox157 FSH, Dupont). To make the basic polyacrylamide gel particle, acentral phase of an aqueous monomer solution containing N-acrylamide(1-20% w/v), a cross-linker (N,N′-bisacrylamide, 0.05-1% w/v), anaccelerator, and ammonium persulfate (1% w/v) was used. An accelerator,(N,N,N′,N′-tetramethylethylenediamine 2% vol %) was added to theoil-phase in order to trigger hydrogel particle polymerization afterdroplet formation.

Several co-monomers were added to the basic gel formulation to addfunctionality. Allyl-amine provided primary amine groups for secondarylabeling after gel formation. FSC of the droplets was modulated byadjusting the refractive index of the gel by adding co-monomers allylacrylate and allyl methacrylate. SSC of the droplets was tuned by addinga colloidal suspension of silica nanoparticles and/or PMMA (poly(methylmethacrylate)) particles (˜100 nm) to the central aqueous phase prior topolymerization.

Stoichiometric labeling of the hydrogel particles was achieved byutilizing co-monomers containing chemically orthogonal side groups(amine, carboxyl, maleimide, epoxide, alkyne, etc.) for secondarylabeling.

Droplets were formed at an average rate of 5 kHz and collected in thefluorocarbon oil phase. After completing polymerization at 50° C. for 30minutes, the resulting hydrogel particles were washed from the oil intoan aqueous solution.

FIG. 6 is a diagram showing an encoding scheme used to create apopulation of similarly-sized beads that can be de-multiplexed usingpassive optical properties, according to some embodiments. Passiveoptical barcoding was achieved via FSC and SSC tuning, and by combiningparticles having unique ratios of FSC/SSC.

FIGS. 7A-7B are characterization plots showing that identically-sizedparticles can be encoded with distinct passive optical properties. Thisallows a multiplexed biochemical assay to be deconvoluted using passiveoptical properties alone. FIG. 7B highlights the inability todistinguish the populations of particles based on fluorescence signalalone, thereby demonstrating biochemical assay multiplexing anddemultiplexing using passive optical properties as a primarydeconvolution variable, according to some embodiments. FIG. 7A showsmultiple synthetic cell populations of the same size, but with distinctpassive optical scatter ratios (FSC/SSC). Distinct surface markers wereconjugated to each subpopulation and co-incubated with FITC conjugatedcognate antibodies. Each biomarker-modified bead population shows anidentical fluorescence profile, but can be deconvoluted based on theirdistinct optical properties to de-multiplex the biochemical assay.

In some embodiments, a composition includes a plurality of hydrogelparticles, with each hydrogel particle from the plurality of hydrogelparticles having substantially the same diameter. The plurality ofhydrogel particles includes a plurality of groups of hydrogel particles,and each group of hydrogel particles from the plurality of groups ofhydrogel particles has a different associated one or more values for apassive optical property (e.g., forward scatter and/or side scatter).

The plurality of hydrogel particles may be included in a mixture, andthe mixture can be configured to be demultiplexed using only passiveoptical properties.

In some embodiments, the plurality of hydrogel particles is included ina mixture, and the mixture is configured to be demultiplexed using (1)passive optical properties and (2) fluorescent properties.

In some embodiments, hydrogel particles from at least one group ofhydrogel particles from the plurality of groups of hydrogel particleshave a refractive index (e.g., an average refractive index or a maximumor minimum refractive index) of greater than about 1.15.

In some embodiments, hydrogel particles from at least one group ofhydrogel particles from the plurality of groups of hydrogel particleshave a refractive index e.g., an average refractive index or a maximumor minimum refractive index) of greater than about 1.3.

In some embodiments, hydrogel particles from at least one group ofhydrogel particles from the plurality of groups of hydrogel particleshave a refractive index of greater than about 1.7.

In some embodiments, each hydrogel particle from the plurality ofhydrogel particles has a diameter of less than about 1000 μm, or of lessthan about 100 μm, or of less than about 10 μm.

In some embodiments, the plurality of hydrogel particles includesnanoparticles.

In some embodiments, at least one hydrogel particle from the pluralityof hydrogel particles is chemically functionalized.

In some embodiments, at least one hydrogel particle from the pluralityof hydrogel particles comprises a free amine group.

In some embodiments, at least one hydrogel particle from the pluralityof hydrogel particles comprises allylamine.

In some embodiments, each hydrogel particle from the plurality ofhydrogel particles is produce by polymerizing a droplet.

In some embodiments, the plurality of hydrogel particles is asubstantially monodisperse population of hydrogel particles.

In some embodiments, a method of performing a multiplexed assay includesassaying a sample using a plurality of optically-encoded hydrogelparticles, deconvoluting the plurality of hydrogel particles using acytometric device and based on passive optical properties of theplurality of hydrogel particles, and determining a plurality ofmeasurements for the sample from a single reaction. Each hydrogelparticle from the plurality of hydrogel particles can be functionalizedwith a different biochemical or chemical target from a set of targets.Alternatively or in addition, each hydrogel particle from the pluralityof hydrogel particles can be functionalized with at least one of: anantigen, a protein, a small molecule, or an antibody.

In some embodiments, each group (from a plurality of groups) of hydrogelparticles from the plurality of hydrogel particles has a differentassociated value for a passive optical property (e.g., forward scatterand/or side scatter).

Example 1 Passive Optical Tuning of Hydrogel Particles

As depicted in FIGS. 4 and 6 , hydrogel particles are tuned in multipledimensions to create distinct populations of beads based on theirpassive optical properties. The beads can be deconvolved usingcombinations of FSC and SSC. An example matching of three primarysubpopulations of white blood cells (lymphocytes, monocytes andgranulocytes (neutrophils)) by tuning passive optical properties,independently of particle size, is shown in FIGS. 8A-8B. For thepurposes of clarity, all particles in the example of FIGS. 8A-8B are thesame diameter. FIG. 8A is a plot of white blood cell (WBC) counts for anactual lysed blood cell population (representing the threesubpopulations), and FIG. 8B shows counts for hydrogel particles withtuned passive optical properties and how they mimic the behavior of thelysed whole blood, with no changes made to the instrument settings(e.g., gains, voltages) between data acquisitions. Some end-userapplications require an assay bead to look optically similar to abiological cell population. The core technology described hereinfacilitates the placement of assay beads precisely on the targetpopulation of interest, while expanding upon multiplexing capabilitiesvia optical encoding.

Example 2 Tuning of Hydrogel Particle Side Scattering

Colloidal silica was added at 12.5%, 6.25%, 3.125% and 0% to the aqueousfraction of the polymer mix and hydrogel particles were formed asdescribed in Example 1. Forward and side scattering data were obtainedusing a flow cytometer. The results showed that side scatter signal(FIG. 9A) increased with higher percentages of encapsulatednanoparticles, while forward scatter signal (FIG. 9B) was generallyunchanged, demonstrating that side scatter can be tuned independently offorward scatter.

Example 3 Tuning of Hydrogel Particle Forward Scattering

In this experiment, the percentage of acrylamide:bis-acrylamide in thehydrogel composition was varied from between 20 and 40% to tune therefractive index of the hydrogel particles as measured by forwardscattering in a flow cytometer. As shown in FIGS. 10A-10C, the forwardscattering increased with increasing percentages ofacrylamide:bis-acrylamide.

All publications, patents, patent applications and other documents citedin this application are hereby incorporated by reference in theirentireties for all purposes to the same extent as if each individualpublication, patent, patent application or other document wereindividually indicated to be incorporated by reference for all purposes.

While various specific embodiments have been illustrated and described,it will be appreciated that various changes can be made withoutdeparting from the spirit and scope of the invention.

While various embodiments of the system, methods and devices have beendescribed above, it should be understood that they have been presentedby way of example only, and not limitation. Where methods and stepsdescribed above indicate certain events occurring in a certain order,those of ordinary skill in the art having the benefit of this disclosurewould recognize that the ordering of certain steps may be modified andsuch modification are in accordance with the variations of theinvention. Additionally, certain of the steps may be performedconcurrently in a parallel process when possible, as well as performedsequentially as described above. The embodiments have been particularlyshown and described, but it will be understood that various changes inform and details may be made. While the embodiments have beenparticularly shown and described, it will be understood that variouschanges in form and details may be made. Although various embodimentshave been described as having particular features and/or combinations ofcomponents, other embodiments are possible having a combination of anyfeatures and/or components from any of embodiments as discussed above.

As used herein, the following terms and expressions are intended to havethe following meanings:

The indefinite articles “a” and “an” and the definite article “the” areintended to include both the singular and the plural, unless the contextin which they are used clearly indicates otherwise.

“At least one” and “one or more” are used interchangeably to mean thatthe article may include one or more than one of the listed elements.

Unless otherwise indicated, it is to be understood that all numbersexpressing quantities, ratios, and numerical properties of ingredients,reaction conditions, and so forth, are contemplated to be able to bemodified in all instances by the term “about”.

As used herein, the terms “about” and “approximately” generally meanplus or minus 10% of the value stated, for example about 250 μm wouldinclude 225 μm to 275 μm, about 1,000 μm would include 900 μm to 1,100μm.

In this disclosure, references to items in the singular should beunderstood to include items in the plural, and vice versa, unlessexplicitly stated otherwise or clear from the context. Grammaticalconjunctions are intended to express any and all disjunctive andconjunctive combinations of conjoined clauses, sentences, words, and thelike, unless otherwise stated or clear from the context. Thus, the term“or” should generally be understood to mean “and/or” and so forth. Theuse of any and all examples, or exemplary language (“e.g.,” “such as,”“including,” or the like) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments or the claims.

1. A composition, comprising: a plurality of hydrogel particles, eachhydrogel particle from the plurality of hydrogel particles havingsubstantially the same diameter, the plurality of hydrogel particlesincluding a plurality of groups of hydrogel particles, each group ofhydrogel particles from the plurality of groups of hydrogel particleshaving a different associated values for a passive optical property. 2.The composition of claim 1, wherein the passive optical property isforward scatter.
 3. The composition of claim 1, wherein the passiveoptical property is side scatter.
 4. The composition of claim 1, whereinthe plurality of hydrogel particles is included in a mixture, and themixture is configured to be demultiplexed using only passive opticalproperties.
 5. The composition of claim 1, wherein the plurality ofhydrogel particles is included in a mixture, and the mixture isconfigured to be demultiplexed using (1) passive optical properties and(2) fluorescent properties.
 6. The composition of claim 1, whereinhydrogel particles from at least one group of hydrogel particles fromthe plurality of groups of hydrogel particles have a refractive index ofgreater than about 1.15.
 7. The composition of claim 1, wherein hydrogelparticles from at least one group of hydrogel particles from theplurality of groups of hydrogel particles have a refractive index ofgreater than about 1.3.
 8. The composition of claim 1, wherein hydrogelparticles from at least one group of hydrogel particles from theplurality of groups of hydrogel particles have a refractive index ofgreater than about 1.7.
 9. The composition of claim 1, wherein eachhydrogel particle from the plurality of hydrogel particles has adiameter of less than about 1000 μm.
 10. The composition of claim 7,wherein each hydrogel particle from the plurality of hydrogel particleshas a diameter of less than about 100 μm.
 11. The composition of claim8, wherein each hydrogel particle from the plurality of hydrogelparticles has a diameter of less than about 10 μm.
 12. The compositionof claim 1, wherein the plurality of hydrogel particles includesnanoparticles.
 13. The composition of claim 1, wherein at least onehydrogel particle from the plurality of hydrogel particles is chemicallyfunctionalized.
 14. The composition of claim 1, wherein at least onehydrogel particle from the plurality of hydrogel particles comprises afree amine group.
 15. The composition of claim 1, wherein at least onehydrogel particle from the plurality of hydrogel particles comprisesallylamine.
 16. The composition of claim 1, wherein each hydrogelparticle from the plurality of hydrogel particles is produce bypolymerizing a droplet.
 17. The composition of claim 1, wherein theplurality of hydrogel particles is a substantially monodispersepopulation of hydrogel particles.